Microbial process for preparing pravastatin

ABSTRACT

The present invention relates to a new microbial process for the preparation of the compound formula (I)  
                 
 
     from a compound of general formula (II)  
                 
 
     wherein R stands for an alkali metal or ammonium ion, by the submerged cultivation of a mold strain able to 6β-hydroxylate a compound of the Formula (II) in aerobic fermentation and by the separation and purification of the product of Formula (I) formed in the course of the bioconversion. The process comprises cultivating a strain of  Mortierella maculata  filamentous mold species that is able to 6β-hydroxylate a compound of the general Formula (II), on a nutrient medium containing assimilable carbon and nitrogen sources and mineral salts and separating the product formed from the fermentation broth, then isolating the compound of formula (I) and purifying the same. Novel strains of  Mortierella maculata  are also disclosed.

FIELD OF THE INVENTION

[0001] The present invention relates to a process for the preparation ofpravastatin, and particularly to a microbial process for the manufactureof pravastatin on an industrial scale.

BACKGROUND OF THE INVENTION

[0002] The highest risk factor of atherosclerosis and especiallycoronary occlusion is the high cholesterol level of the plasma. In thelast two decades 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC.1.1.1.34) as the rate limiting key enzyme of the cholesterolbiosynthesis was extensively examined. Pravastatin, a compound ofFormula I,

[0003] and other related compounds (compactin, mevinolin, simvastatin)are the competitive inhibitors of the HMG-CoA reductase enzyme [A. Endoet al., J. Antibiot. 29, 1346-1348 (1976); A. Endo et al., FEBS Lett.72, 323-326 (1976); C. H. Kuo et al., J. Org. Chem. 48, 1991 (1983)].

[0004] Pravastatin was first isolated by M. Tanaka et al. (unpublishedresults) from the urine of a dog during the examination of the compactinmetabolism (Arai, M. et al., Sankyo Kenkyusyo Nenpo, 40, 1-38, 1988).Currently pravastatin is a cholesterol lowering agent with the mostadvantageous action mechanism in the therapy. Its most importantcharacter is tissue selectivity, i.e., it inhibits the cholesterolsynthesis at the two main sites of the cholesterogenesis, such as in theliver and in the small intestine, while in other organs theintracellular enzyme limiting effect is hardly detectable, At the sametime the cholesterol biosynthesis limiting effect of mevinolin andsimvastatin is significant in most of the organs (T. Koga et al.,Biochim. Biophys. Acta, 1045, 115-120, 1990).

[0005] Pravastatin essentially differs in chemical structure frommevinolin and simvastatin which have more lipophilic character. In thecase of the latter compounds the substituent connected to the C-1 carbonatom of the hexahydronaphthalene skeleton is ended in a 6-memberedlactone ring, while in the case of pravastatin, instead of the lactonering, the biologically active, opened dihydroxy acid sodium salt form ispresent. Another important structural difference is that instead of themethyl group of mevinolin and simvastatin at the C-6-position of thehexahydronaphthalene ring, a hydroxyl group can be found in pravastatin,which results in a further increase in its hydrophilic character.

[0006] As a result of the above structural differences pravastatin isable to penetrate through the lipophilic membrane of the peripheralcells only to a minimal extent (A. T. M., Serajuddin et al., J. Pharm.Sci. 80, 830-834, 1991).

[0007] Industrial production of pravastatin can be achieved by twofermentation processes. In the first, microbiological stage compactin isprepared, then in the course of a second fermentation the sodium salt ofcompactin acid as a substrate is converted to pravastatin by microbialhydroxylation at the 6β-position.

[0008] According to published patents, the microbial hydroxylation ofcompactin can be accomplished to various extents with mold speciesbelonging to different genera, and with filamentous bacteria belongingto the Nocardia genus, with Actinomadura and Streptomyces genera(Belgian patent specification No. 895090, Japanese patent specificationNo. 5,810,572, U.S. Pat. Nos. 4,537,859 and 4,346,227 and publishedEuropean patent application No. 0605230). The bioconversion of compactinsubstrate was published in a 500 μg/ml concentration using filamentousmolds such as Mucor hiemalis, Syncephalastrum nigricans, Cunninghamellaechinulata and in 2000-4000 μg/ml with Nocardia, Actinomodura andStreptomyces strains belonging to the prokaryotes.

[0009] A general problem experienced in the cases of manufacturing thepravastatin with filamentous molds is that due to the antifungal effectof compactin, the microorganisms are not able to tolerate the compactinsubstrate fed to the culture even at low concentrations (Serizawa etal., J. Antibiotics, 36, 887-891, 1983). The cell toxicity of thissubstrate was also observed in the hydroxylation with Streptomycescarbophilus extensively studied by Japanese researchers (M. Hosobuchi etal., Biotechnology and Bioengineering, 42, 815-820, 1993).

[0010] Japanese authors tried to improve the hydroxylating ability ofthe Streptomyces carbophilus strain with recombinant DNA techniques. Acytochrome P-450 monooxygenase system is needed for the hydroxylation ofcompactin (Matsuoka et al., Eur. J. Biochem. 184, 707-713, 1989).However, according to the authors, in the bacterial cytochrome P-450monooxygenase system not one but several proteins act in the electrontransport, which aggravate the application of the DNA techniques.Development of a cost-effective microbiological hydroxylation method forthe manufacture of pravastatin is an extremely difficult, complex task.

[0011] The aim of the present invention is to elaborate a new microbialprocess for the preparation of pravastatin from compactin in industrialscale, which would produce pravastatin at more advantageous conditionsthan those previously known. During our research work, above all wetried to find a microorganism strain with a hydroxylase enzyme that canbe adapted for the microbial transformation of compactin to pravastatinin a high concentration.

SUMMARY OF THE INVENTION

[0012] The present invention relates to a microbial process for thepreparation of the compound of formula (I)

[0013] from a substrate compound of formula (II),

[0014] wherein R stands for an alkali metal or ammonium ion,

[0015] comprising the steps of (a) cultivating a strain of Mortierellamaculata filamentous mold species able to 6β-hydroxylate a compound offormula (II) on a nutrient medium containing assimilable carbon- andnitrogen sources and mineral salts, (b) feeding the substrate to betransformed into the developed culture of Mortierella maculata, (c)fermenting the substrate until the end of bioconversion, (d) separatingthe compound of formula (I) from the culture broth, and (e) isolatingthe compound of formula (I).

[0016] The present invention also relates to a biologically pure cultureof the Mortierella maculata n. sp. E-97 strain deposited at the NationalCollection of Agricultural and Industrial Microorganisms, Budapest,Hungary under accession number NCAIM(P)F 001266 and a biologically pureculture of its mutant, the Mortierella maculata n. sp. E-97/15/13 straindeposited at the National Collection of Agricultural and IndustrialMicroorganisms, Budapest, Hungary under accession number NCAIM(P)F001267.

BRIEF DESCRIPTION OF THE DRAWING

[0017]FIG. 1 is an illustration of the physical characteristics ofMortierella maculata n. sp. E-97.

DETAILED DESCRIPTION OF THE INVENTION

[0018] In the course of our screening program, which covered about 5500prokaryotic and eukaryotic strains, 23 microorganisms were selected,which were able to hydroxylate compactin in opposition. Among thesestrains a filamentous mold proved to be more appropriate for theproduction of pravastatin due to its higher resistance against compactinas compared to the strains known from published patents. According tothe taxonomic investigation, this strain proved to be a newrepresentative of the species belonging to the Mortierella genus(Mortierella maculata n. sp.). From the selected molds a new strain wasisolated on the one hand by the application of the mutation-selectionmethods, and on the other hand by the induction of the hydroxylaseenzyme of the strain, which one was able to hydroxylate the compactinsubstrate to pravastatin in a higher concentration than published sofar. As mutagenic agents, physical and chemical mutagens were applied(UV irradiation, methyl methane sulfonate,N-methyl-N′-nitro-N-nitrosoguanidine). After the mutagenic treatments,in order to prepare haploid cells, the spore suspension was spread onbenomyl-containing agar plates, then in order to induce the hydroxylaseenzyme the developed colonies were inoculated onto 100 μg/ml8-de-(2-methyl-butyryl)-compactin-containing or compactin-containingagar plates. By the application of these methods a mutant strain wasprepared from the new strain that is able to convert compactin topravastatin to a significantly higher extent than the parent strain.

[0019] In the course of the optimizing experiments we determined thecomposition of the most beneficial inoculum, and the most advantageousbioconversion media for the compactin hydroxylation, as well as theoptimal method for the repeated feeding of compactin in a highconcentration.

[0020] Consequently, this invention is based on the recognition that theE-97 and E-97/15/13 designated strains of the isolated mold namedMortierella maculata, which were deposited under accession numbers ofNCAIM(P)F 001266 and NCAIM(P)F 001267 respectively, at the NationalCollection of Agricultural and Industrial Microorganisms (Department ofMicrobiology and Biotechnology, University of Horticulture and the FoodIndustry Budapest), under appropriate fermentation conditions are ableto manufacture pravastatin to a high extent, while the undesired relatedcompounds such as the acid forms of 6α-hydroxy-compactin,2α-hydroxy-compactin, 8-de-(2-methyl-butyryl)-compactin,3α,5β-dihydroxy-5,6-dihydro-isocompactin, 8α,β-hydroxy-compactin and thehydroxylated derivatives at positions 2 and 3 of the 2-methyl-butyrylside chain of compactin are obtained only in small or trace amountsduring the bioconversion. Thus, these strains are especially appropriatefor manufacturing pravastatin in an industrial scale.

[0021] Taking into account that the economical manufacture of the activeingredient on an industrial scale is a function of the compactinsubstrate concentration, it is important to have a strain that is ableto tolerate high compactin and pravastatin concentrations. Consequently,a further important part of the invention is the recognition that thehydroxylating ability of the original mold isolate can be improved bythe application of mutation-selection and enzyme induction methods and,furthermore, that by the development of an appropriate method forsubstrate feeding the hydroxylation of large quantities of compactin topravastatin can be executed in a single procedure. In conclusion, thenew mutant strain designated as Mortierella maculata n. sp. E-97/15/13is especially appropriate for the manufacture of pravastatin.

[0022] Taxonomic features of the isolated new mold species comparing itto the most important diagnostic attributes of the known Mortierellaspecies are summarized below.

[0023] Taxonomic Description of the Holotype Strain Mortierella maculataNov. Spec. E-97

[0024] On starch-casein-malt extract-agar media the aerial mycelium iswell developed (more than 10 μm thick covering layer over the substratemycelium). At the beginning it appears as a tightly woven white web ofhyphae, in which later yellowish sporulating spots with a few mmdiameter sparsely appear (new name “maculatus” refers to the above:spotted). This yellowish coloration can sometimes occupy the largercontinuous surfaces of the aerial web. The color of the substratemycelium is on Czapek-, bloody-Czapek-, tyrosine-, starch-casein-, maltextract-, etc. agar media mostly colorless or light yellowish. The colorof the substrate mycelial web is light reddish on yeastextract-glucose-peptone medium. Production of diffusible and solublepigments on the above listed media is not experienced, or only rarely aninsignificant yellowish coloration occurs on these media. Colonies ofstrain E-97, due to their volatile oil production and similarly to manyother species of Mortierella, (except to species of section Isabellina),can exude a very characteristic strong scent.

[0025] Sporangiophores, designated by reference numerals 1-7 in FIG. 1,frequently develop locally on the aerial hyphae (but less on thesubstrate ones) in great numbers at very different distances from eachother. They are not branching, but are mostly straight or curved. Theirlength is generally between 60-80 μm. The starting point in theoverwhelming majority of cases is a more or less short but stronglyswollen hyphal section of the aerial web, from which they are separatedby walls. Sporangiophores themselves can be also swollen (sometimesstrongly), as shown by reference numeral 6, but in the direction of thesporangium they gradually narrow, from 5.0-9.0 μm to 1.0-2.0 μm. It isan important taxonomic character that below the sporangiophores theynever broaden out (see reference numeral 8).

[0026] Sporangia are spherical; in some cases slightly flattenedspheres. Their diameter is about 6.0-17.0 μm, relatively small comparedto the measures of sporangia of other Mortierella species. Sporangia maycontain many spores, but sporangia bearing only one spore also exist.The spores 9 are cylindrical or less oval. Their size is 3.0-5.0×1.5-2.0μm. Within the individual spores one or two small dark sphericaloil-drops 10 may be present. Due to the very easy disintegration of thewall of sporangia, in wet surroundings the spores will quickly bescattered. After the disintegration of the sporangium, sometimes at theend of the sporangiospores, a fine pitchfork-like “collar” and a veryshort rudimental (and not typical) columella can be observed. Gemmae15-28 that are spherical or cylindrical may occur on most differentdiagnostic media. Usually the size is 10-25 μm. In the cultures chainsof spherical gemmae 13, budding cells, intercalated gemmae 15-23, hyphalassociations of particular spiral-growth of one hypha around the other11, anastomotic-like structures and giant cells, etc. can also be found.In the aerial mycelium also large (50-250 pm diameter) very dense hyphalwebs 14 can be seen but without the presence of detectable zygotes.

[0027] Cultures of strain E-97 are able to reduce nitrates to nitrites,do not hydrolyze starch, esculin, arginine or gelatine but hydrolyzeTween polysorbates and do not decompose paraffin hydrocarbons. Thecultures of strain E-97 have urease activity show a good growth betweenpH 7.0 and 9.0 tolerate a maximum 2% NaCl. The effect of xanthine,hypoxanthine, lecithin, tyrosine and adenine are negative. A strong acidproduction of the cultures has been detected from glucose, fructose,glycerine and galactose, but very weak or no production from xylose,arabinose, raffinose, sorbitol, inositol, inulin, etc. Weak growth isdetected on pyruvate and acetate but no growth could be found withbenzoate, salicylate, citrate, lactate, succinate, tartarate andmalonate. A good growth was observed with glucose and fructose as solecarbon-sources in the medium. Utilization tests with xylose, arabinose,rhamnose, sucrose, raffinose, mannitol and inositol proved to benegative. The cultures do not decompose cellulose.

[0028] Systematic position: Strain E-97 belongs to the familyMortierellaceae and it is a typical member of the genus Mortierella:sporangia contain generally many spores, columella is extremely reduced,gemmae are frequently present, the occurrence of zygotes has not beendetected and the colonies exude a very characteristic strong scent.Within the genus Mortierella, strain E-97 is a typical representative ofthe “Section Alpina.” The latter can be characterized by very shortnon-branching sporangiophores (maximal length to 200 μm), and minutesporangia (Zycha, H. und Siepmann, R, Mucorales. Eine Beschreibung allerGattungen und Arten dieser Pilzgruppe. D-3301 Lehre, Verl. von J.Cramer. 1969). Among the members of the section Alpina, strain E-97shows the greatest similarity to the species M. thaxteri Björling 1936and M. renispora Dixon-Stewart 1932. However, the data in the Table Iclearly show the differences in diagnostic properties of strain E-97 andof these two species. Accordingly, as a new species herewith weintroduce the strain under the name Mortierella maculata nov. spec.E-97. TABLE 1 A comparison of the holotype strain E-97 of Mortierellamaculata n. sp. with the species M. renispora and M. thaxteri on thebasis of key diagnostic properties Mortierella renispora Mortierellastrain E-97 Mortierella thaxteri Origin of Ordinary, however theLaterally from (swollen or Laterally from swollen separatedsporangiophores hyphae are wider than the normal) aerial hyphae or notsegments of aerial hyphae or not regular ones, laterally from separatedsections of substrate separated segments of substrate hyphae broadenedswollen hyphae. hyphae. Shape and size of Gradually decreasing towardsMostly straight or curved, not Length about 60-90 μm. Width at thesporangiophores the top from 10 μm to 3 μm. branching. Width graduallystarting point is about 5-7 μm, at the tip Length is about 200 μm.decreasing towards the tip: it is reduced to 1.5-2 μm. Immediately from5-9 μm to 1.5-2.5 μm. under the sporangium broadened out Length is about60-80 μm. At the tip never broadened. Shape and size of Colorless,diameter is 25 μm. Mostly spherical (6-17 μm Spherical (12-20 μm diam.).They sporangia diam.) but rarely less flattened. contain many spores buton certain Generally contain many media there is also only one spore-spores, rarely one spore. bearing sporangra. Wall (membrane) Spreadingmembrane. After Disintegrating. A pitchfork- Disintegrating, a minutebackward - of the sporangia disintegration remains a like collarremains. bending collar remains. collar-like structure. Shape and sizeof Roughly kidney-shaped, Cylindrical. Length (3-5 μm) Ellipsoidalhyaline spores of 3.5-4 × 1.5-2 μm) the spores hyaline structures, sizesare can doubly exceed the width dimension. 2 × 4 μm. (1.5-2 μm). GemmaeOccur on the most different Frequent on very different Intercalary, ovalgemmae (10-14 μm) in media media, mostly in the aerial the substratemycelium. mycelium. Spherical or elongated (10-25 μm). Zygotes Frequenton all diagnostic Not detected. Not observed. media. Diameter togetherwith the covering hyphae is about 500 μm, without them about 30 μm.Dense foci of the Large densely woven hyphal In old cultures large(100-125 μm hyphal web foci (50-250 μm) frequent but diam)yellowish-grey dense hyphal without zygotes. webs, without zygote. Colorand habit of Loose, always white hyphal White with yellowish spots. Atfirst spider's web-like, later more the aerial mycelium web dense.

[0029] In the process for the preparation of pravastatin according tothe present invention, preferably the culture of the mold straindesignated as Mortierella maculata n. sp. E-97 or its mutant designatedas E-97/15/13 is used. The selected strain is highly advantageous due toits fast growth. As a carbon source it easily utilizes glucose,glycerine, fructose, or galactose. As a nitrogen source yeast extract,peptone, casein, meat extract, soybean meal, corn steep liquor, sodiumnitrate, or ammonium sulfate can be used.

[0030] In the culture media used for the production of pravastatinbesides the above carbon and nitrogen sources mineral salts, e.g.,potassium dihydrogen phosphate, magnesium chloride, magnesium sulfate,trace elements (ferrous, manganous salts), amino acids and antifoamingagents, can be present.

[0031] According to a preferred embodiment of the present invention, thespore suspension having been prepared from the slant agar culture of theMortierella maculata n. sp. designated as E-97 strain or its mutant[NCAIM(P)F 001267] designated as E-97/15/13, is seeded into an inoculummedium; then 10% of the inoculum culture, which is cultivated for 3 daysat about 25-30° C., preferably at about 24-28° C., most preferably atabout 28° C., is transferred into the bioconversion medium. Then it isincubated for 4 days at about 25-28° C., preferably at about 28° C.,then glucose and the sodium salt of compactin acid are fed into theculture. Depending on the concentration of the fed compactin substrate,the cultivation is continued for 2-12 days further under aerobicconditions, while the pH is maintained between 5.5 and 7.5, preferablyat 7.0. The bioconversion is done under stirred and aerated conditions,when the air flow rate is 0.2 vvm, the spinning rate of the stirrer is400/min.

[0032] In the course of the fermentation the bioconversion of compactinsubstrate was followed by a high pressure liquid chromatographic method(HPLC). According to this method, the sample of the broth is dilutedtwofold with methanol and centrifuged, and the supernatant is used forthe HPLC analysis under the following parameters: Waters analytical HPLCequipment; column: Nucleosil C₁₈ 10 μm; detection wavelength: 238 nm;injection volume: 20 μl, flow rate: 1 ml/min; gradient elution is used,eluents: A=0.05% aqueous solution of phosphoric acid, B=acetonitrile.

[0033] Elution gradient: Time (min) Eluent A (%) Eluent B (%) 0 70 30 200 100 25 0 100 25.1 70 30 35 70 30

[0034] Approximate retention times: pravastatin 8.6-9.0 min; compactinacid 11.6-12.0 min; pravastatin lactone 15.0-15.5 min, compactin16.5-17.0 min.

[0035] For the production of pravastatin the aqueous solution of thesodium salt of compactin acid is added at the 96th hour of thecultivation. For this procedure the substrate is prepared in solid formas follows. Compactin lactone is hydrolyzed in a 0.2M sodium hydroxidesolution for 2 hours at 40° C., then the pH of the reaction mixture isadjusted to 7.5 by hydrochloric acid and the neutralized solution islayered on a Diaion HP-20 adsorbent column; the sodium chloride formedduring the neutralization is eliminated by aqueous washing of thecolumn, and then the sodium salt of the compactin acid is eluted fromthe column by 50% aqueous acetone. Thereafter the eluate is distilled invacuum and the aqueous residue is lyophilized. After neutralization theaqueous solution of the alkaline hydrolysate of compactin can also bedirectly used as substrate. In this case the compactin acid sodium saltcontent of the hydrolysate is measured by HPLC, and the solution is keptat −20° C. until being applied.

[0036] The higher the broth pH reached by the fourth day of thefermentation, the more advantageous for the hydroxylation of thecompactin substrate. Feeding of the compactin substrate is allowed to bestarted when the pH of the broth exceeds 6.3. At the 4th day of thefermentation as much of the sterile filtered aqueous solution ofcompactin acid sodium salt is added as needed to reach the 500 μg/mlconcentration. Glucose is also fed to the culture from its 50% solutionsterilized at 121° C. for 25 minutes as follows: if the pH of the brothis higher than 6.7 value, 1% glucose is added related to the volume ofthe broth, while if the pH is within the 6.3-6.7 range the quantity ofthe glucose fed is 0.5%. Compactin acid sodium salt is consumed from thebroth after 24 hours, its transformation is analyzed by HPLCmeasurement. In this case, for each ml of the broth another 500 μg ofcompactin is added. Besides the compactin substrate, glucose is also fedas described above. Morphology of the 120 hour culture is characterizedby the small pellet growth (diameter of the pellet; 0.5-3.0 mm). After24 hours the second dose of substrate is also consumed from the broth,thus a further portion of compactin acid sodium salt producing a 500μg/ml concentration of it in the whole broth is added parallel with theglucose feeding dependent on the pH value of the broth. From the 4th dayof the fermentation the substrate and the glucose feeding is repeated indaily frequency as it is written before until the 17th-18th day of thefermentation.

[0037] For the recovery of the product from the broth, it isadvantageous to take into consideration the fact that during thebioconversion pravastatin is formed in its acidic form, thus it can beisolated from the filtrate of the broth by its adsorption on an anionexchange resin column. For the isolation of the product it isadvantageous to use a strongly basic anion exchange resin which is apolystyrene-divinylbenzene polymer carrying quaternary ammonium activegroups. The product can be adsorbed directly from the filtrate of thebroth by mixing the anion exchange resin being in hydroxyl form into it.The product being adsorbed on the ion exchange resin can be eluted fromthe column by acetic acid or a sodium chloride-containing acetone-watermixture, preferably 1% sodium chloride containing acetone-water (1:1)mixture. Pravastatin-containing fractions are combined and the acetonebeing in the eluate is distilled off in vacuum. The pH of theconcentrate is adjusted with 15% sulfuric acid into the range of 3.5-4.0and the aqueous solution is extracted by ethyl acetate. The ethylacetate extract is washed with water and dried with anhydrous sodiumsulphate. Then the lactone derivative is prepared from pravastatin. Thelactone ring closure is carried out in dried ethyl acetate solution atroom temperature, under continuous stirring by inducing the lactoneformation with trifluoroacetic acid being present in catalytic quantity.The transformation procedure is checked by thin layer chromatographicanalysis (TLC). After finishing the lactone formation the ethyl acetatesolution is washed at first with 5% aqueous sodium hydrogen carbonatesolution and then with water, then it is dried with anhydrous sodiumsulfate and evaporated in vacuum. The evaporated residue is treated inacetone solution with charcoal, then evaporated again and recrystallizedfrom a 1-4 carbon atom-containing aliphatic alcohol, preferably fromethanol. The evaporation residue of the recrystallization mother liquoris purified with silica gel column chromatography applying the mixtureof ethyl acetate-n-hexane with gradually increasing ethyl acetatecontent as the eluent.

[0038] From the pravastatin lactone obtained after recrystallization andchromatographic purification pravastatin is prepared by hydrolysis atroom temperature in acetone with equivalent quantity of sodiumhydroxide. When the pravastatin sodium salt formation is completed, thereaction mixture is diluted with water and neutralized, and the acetonecontent is distilled in vacuum. Pravastatin is adsorbed from theobtained aqueous residue on a Diaion HP-20 resin-containing column,washed with deionized water and eluted from the column with anacetone-deionized water mixture. Then the pravastatin containingfractions are combined, the acetone content is distilled off and afterthe lyophilization of aqueous residue pravastatin can be obtained inhigh purity, which can be recrystallized from an ethyl acetate-ethanolmixture.

[0039] In the course of the procedure the whole quantity of pravastatincan be adsorbed. During the lactone closure of pravastatin3α-hydroxy-iso-compactin and other by-products can also be formed.Although these latter reactions decrease the yield of the isolation butthose compounds can be separated by the above-described purificationmethod and consequently, pravastatin can be manufactured this way in apharmaceutically acceptable quality.

[0040] After finishing the bioconversion pravastatin can be extractedeither from the fermentation broth or from the filtrate obtained afterthe separation of the filamentous mold cells. Filamentous mold cells canbe eliminated either by filtration or centrifugation; however, it isadvantageous especially on an industrial scale to make a whole brothextraction. Before extraction the pH of either the fermentation broth orthe filtrate of the broth is adjusted to 3.5-3.7 with a mineral acidpreferably with diluted sulfuric acid. The extraction is done with anester of acetic acid and a 24 carbon atom containing aliphatic alcohol,preferably with ethyl acetate or isobutyl acetate. Extraction stepsshould be done very quickly in order to avoid the formation of thelactone derivative from pravastatin at acidic pH.

[0041] From the organic solvent extract the pravastatin in acid form canbe transferred as the sodium salt into the aqueous phase. For example,from an ethyl acetate extract pravastatin can be extracted by 1/10 and1/20 volume ratio of 5% sodium hydrogen carbonate or weakly alkalinewater (pH 7.5-8.0). It was found that pravastatin can be recovered in apure form from the above-obtained alkaline aqueous extract by columnchromatography with the application of a non-ionic adsorption resin. Anadvantageous method is to first remove the solvent dissolved in theaqueous phase by vacuum distillation from the alkaline aqueous extract,and then the aqueous extract is loaded on a Diaion HP-20 column.

[0042] Pravastatin sodium salt being adsorbed on the column is purifiedby elution increasing gradually the acetone content of the aqueoussolutions, then the pravastatin-containing main fractions are combinedand concentrated in vacuum. The aqueous concentrate is purified furtherby chromatography on another Diaion HP-20 column, obtaining an eluatecontaining pure pravastatin, from which after clarification withcharcoal and lyophilization pravastatin can be obtained in apharmaceutically acceptable quality.

[0043] This isolation procedure consists of fewer stages than theprevious one, since the lactone formation of pravastatin and itshydrolysis are not involved in the procedure. During the isolationpravastatin is exposed to acidic condition for only a limited time,under which it is less stable than in neutral or alkaline solutions,consequently, during this isolation procedure artefacts are practicallynot formed.

[0044] Furthermore, it was found that the chromatography on SephadexLH-20 Dextran gel (hydroxypropylated derivative) is advantageously usedfor purifying pravastatin. By application of this method pravastatinexceeding the purity of 99.5% (measured by HPLC) can be produced.

[0045] In the course of our experiments it has been recognized that fromthe organic solvent extract, preferably from the ethyl acetate orisobutyl acetate extract of the broth or the broth nitrate of thefilamentous mold or the filamentous bacteria strains among them theMortierella maculata n. sp. strain able to 6β-hydroxylate a compound ofgeneral formula (II), pravastatin can be precipitated as a crystallinesalt with secondary amines. Further it was found that for the saltformation several secondary amines containing alkyl, cycloalkyl-,aralkyl- or aryl-substituents are appropriate. Expediently non-toxicsecondary amines were selected among them, e.g., dioctylamine,dicyclohexylamine, dibenzylamine. The isolation of the organic secondaryamine salt intermediates, e.g., the dibenzylamine salt was carried outby adding dibenzylamine in 1.5 equivalent quantity related to thepravastatin content of the extract, then the extract is concentrated byvacuum distillation to 5% of its original volume, then another quantityof dibenzylamine is added into the concentrate in 0.2 equivalent ratio.The crystalline dibenzylamine salt is precipitated from the concentrate.The crystalline crude product is filtered and dried in vacuum. Then itis clarified with charcoal and recrystallized in acetone.

[0046] In the procedure mentioned earlier in which the organic solventextraction and the reextraction at alkaline pH are involved, theisolation method based on the secondary amine salt formation can be usedalso for the replacement of the column chromatographic purification. Inthis case it is advantageous to precipitate the pravastatindibenzylamine salt from the isobutyl acetate extract obtained after theacidification of the alkaline aqueous extract.

[0047] Pravastatin organic secondary amine salts can be transformed topravastatin by sodium hydroxide or a sodium alkoxide, preferably sodiumethoxide.

[0048] The transformation is detailed in the case of pravastatindibenzylamine salt. The recrystallized dibenzylamine salt is suspendedin an isobutyl acetate-water mixture, then equivalent quantity of sodiumhydroxide is added in aqueous solution to the suspension by maintainingunder stirring the pH in the range of 8.0-8.5. After disappearance ofthe suspension the phases are separated and the pravastatin-containingaqueous solution is washed twice with isobutyl acetate. The aqueoussolution is clarified with activated carbon and lyophilized yieldingpravastatin in a pharmaceutically acceptable quality.

[0049] One preferred method for the transformation of pravastatindibenzylamine salt to pravastatin is to suspend the recrystallizeddibenzylamine salt in ethanol, then equivalent quantity or small excessof sodium ethoxide is added under stirring to the suspension, then thereaction mixture is concentrated in vacuum and by adding acetone thepravastatin is precipitated in crystalline form from the concentrate.

[0050] Another preferred method for the transformation of pravastatindibenzylamine salt to pravastatin is to dissolve the recrystallizeddibenzylamine salt in ethyl acetate-ethanol mixture and by addingequivalent quantity or small excess of sodium hydroxide in ethanol tothe solution pravastatin is precipitated.

[0051] The isolation of pravastatin via a secondary amine saltintermediate is a simpler procedure than any previously known isolationprocedures. During the procedure artifacts are not formed, and theseparation of pravastatin from the by-products of the bioconversion andfrom the various metabolic products biosynthesized by the hydroxylatingmicroorganism can be solved without the application of anychromatographic methods.

[0052] The structures of pravastatin, pravastatin lactone and theisolated secondary amine salts of pravastatin have been proven by UV,IR, ¹H-NMR, ¹³C-NMR and mass spectroscopic methods.

EXAMPLES

[0053] The invention will be more fully described and understood withreference to the following examples, which are given by way ofillustration and are not intended to limit the scope of the invention inany way.

Example 1

[0054] A spore suspension was prepared with 5 ml of a 0.9% sodiumchloride solution obtained from a 7-10 day old, malt extract-yeastextract agar slant culture of Mortierella maculata nov. spec. E-97[NCAIM(P)F 001266] strain able to 6β-hydroxylate compactin and thesuspension was used to inoculate 100 ml inoculum medium PI sterilized ina 500 ml Erlenmeyer flask.

[0055] Composition of the medium PI: glucose 50 g soybean meal 20 g in1000 ml tap water.

[0056] Before the sterilization the pH of the medium was adjusted to7.0, then it was sterilized at 121° C. for 25 min. The culture wasshaken on a rotary shaker (250 rpm, 2.5 cm amplitude) for 3 days at 28°C., then 10 ml of the obtained culture was transferred into 100-100 mlbioconversion media MU/4 sterilized in 500 ml Erlenmeyer flask for 25min at 121° C.

[0057] Composition of the medium MU/4: glucose  40 g soybean meal  20 gcasein-peptone   1 g asparagine   2 g potassium dihydrogen phosphate 0.5g in 1000 ml tap-water.

[0058] Before the sterilization the pH of the medium was adjusted to7.0, then it was sterilized at 121° C. for 25 min.

[0059] Flasks were shaken on a rotary shaker (250 rpm, 2.5 cm amplitude)for 4 days at 25° C., then 50-50 mg compactin substrate (compactin acidsodium salt) was added in sterile-filtered aqueous form into thecultures, then the cultivation was continued. Similarly, at the 5th dayanother 50-50 mg compactin acid sodium salt was added into the moldcultures, and the fermentation was continued for a further 24 hours. Thepravastatin content of the broth was determined by HPLC. Thefermentation was continued for 168 hours. At the end of thebioconversion the average pravastatin concentration of the fermentationbroth was 620 μg/ml.

Example 2

[0060] In a laboratory scale fermenter with 5 liters working volume aMU/S bioconversion culture medium is prepared, the components of theculture medium are added corresponding to 5 liters, volume but it wasloaded up only to 4.5 liters, then it was sterilized for 45 min at 121°C. and seeded with 500 ml of the inoculum culture made according to theExample 1.

[0061] Composition of medium MU/8: glucose  20 g glycerine  20 g soybeanmeal  20 g peptone   5 g potassium dihydrogen phosphate 0.5 gpolypropyleneglycol 2000   1 g in 1000 ml tap water.

[0062] Before sterilization the pH of the medium was adjusted to 7.0value.

[0063] The fermentation was carried out at 28° C., with a stirring rateof 400 rpm and with an aeration rate from bottom direction 60liters/hour for 4 days. At the 2nd day after the transfer the culturestarted to foam heavily, which can be decreased by the addition offurther polypropyleneglycol 2000. At the beginning of the fermentation(16-20 hours) the pH decreased from the initial value of 6.5 to 5.0-5.5,then from the 3rd day it started to increase and reached 6.3-7.5 by the4th day. The feeding of the compactin substrate is allowed to be startedif the pH of the broth is above 6.3. At the 4th day of the fermentation2.5 g compactin substrate is added in sterile filtered aqueous solution.Calculated for the volume of the broth 0.5-1.0% glucose was added intothe culture depending on the pH in the form of 50% solution sterilizedat 121° C. for 25 minutes in parallel with the substrate feeding. After24 hours the compactin substrate is consumed from the culture, which isdetected by HPLC from the samples taken from the fermenter. In this caseanother 2.5 g compactin substrate and glucose were added as describedabove, and the bioconversion was continued for 24 hours further when thesubstrate was converted to pravastatin.

[0064] After finishing the fermentation, 5.1 liters broth containing 630μg/ml pravastatin were filtered on a filter cloth. Two liters water wereadded to the separated mycelium, then the mycelium suspension wasstirred for one hour and filtered. These two filtrates were combined andpassed through with a flow rate of 500 ml/hour on a column containing138 g (250 ml) Dowex Al 400 (OH) resin (diameter of the column 3.4 cm,height of the resin bed: 28 cm), then the resin bed was washed with 300ml deionized water. Subsequently, the elution from the resin was carriedout by 1 liter acetone-water (1:1) mixture containing 10 g sodiumchloride. The volume of the fractions was 100 ml each. The eluate wasanalyzed by the following thin layer chromatographic (TLC) method:adsorbent: Kieselgel 60 F 254 DC (Merck) aluminum foil; developingsolvent: acetone-benzene-acetic acid (50:50:3) mixture; detection: withphosphomolybdic acid reagent. The R_(f) value of pravastatin is 0.5.Fractions containing the product were combined and the acetone wasdistilled off in vacuum. The pH of the 400 ml concentrate was adjustedto 3.5-4.0 by 15% sulfuric acid, then it was extracted three times by150 ml ethyl acetate. The ethyl acetate extracts were combined and driedwith anhydrous sodium sulfate. Subsequently, pravastatin lactone wasprepared from pravastatin acid by adding at room temperature undercontinuous stirring trifluoroacetic acid in catalytic amount. Theformation of pravastatin lactone was controlled by TLC (the R_(f) valueof pravastatin lactone in the above TLC system is 0.7). After thecompletion of the lactone formation, the ethyl acetate was washed with2×50 ml 5% aqueous sodium hydrogen carbonate solution, then washed with50 ml water, dried with anhydrous sodium sulfate and evaporated invacuum. The evaporation residue obtained in a quantity of 3 g wasdissolved in 100 ml acetone and clarified with 0.3 g charcoal. Then thecharcoal was filtered off and the acetone was evaporated in vacuum. Thecrude product obtained was crystallized from 20 ml ethanol. Precipitatedcrystalline pravastatin lactone was filtered off, and washed on thefilter with 30 ml n-hexane and dried at room temperature in vacuum. Inthis way 1.5 g chromatographically pure pravastatin lactone wasobtained. Melting point 140-142° C., [α]_(D)=+194° (c=0.5, methanol).The mother liquor of the crystallization was evaporated in vacuum and1.2 g evaporation residue is obtained, which was chromatographed on 24 gKieselgel 60 adsorbent containing column (diameter of the column: 1.6cm, height of the bed: 20 cm). The crude product dissolved in 5 mlbenzene was layered on the column. For elution mixtures of ethylacetate-n-hexane were used in which the ethyl acetate content wasgradually increased. Pravastatin lactone can be eluted from the columnwith the mixture of 60% ethyl acetate-40% n-hexane. Fractions werecontrolled by TLC using the mixture of ethyl acetate-n-hexane (9:1) asthe developing solvent. The pravastatin lactone-containing fractionswere combined and evaporated in vacuum. According to this method 0.3 gpure product is obtained, its quality identical with that of thepravastatin lactone obtained by crystallization.

[0065] The two pravastatin lactone batches were combined and the sodiumsalt was prepared according to the following method: 1.8 g pravastatinlactone was dissolved in 20 ml acetone and under stirring 4.5 ml of 1Maqueous sodium hydroxide was added, then the solution was stirred forhalf an hour at room temperature. When the salt formation was completed,20 ml water was added into the mixture and the solution was neutralized,then the acetone was evaporated in vacuum. The aqueous concentrate waschromatographed on a column filled with 150 ml Diaion HP 20 resin(diameter of the column: 2.6 cm, height of the bed: 30 cm). As theeluting agent mixtures of acetone-deionized water were used, where theconcentration of the acetone was increased in 5% steps. Pravastatin canbe eluted from the column by a 15% acetone containing acetone-deionizedwater mixture. Fractions were analyzed by TLC. Fractions containing theproduct are combined and acetone was evaporated in vacuum. Bylyophilization of the aqueous residue 1.3 g pravastatin was obtained.The chromatographically pure product was crystallized from a mixture ofethanol and ethyl acetate.

[0066] Melting point: 170-173° C. (decomp.)

[0067] [α]^(D) ₂₀=+156°, (c=0.5, in water).

[0068] Ultraviolet absorption spectrum (20 μg/ml, in methanol):λ_(max)=231, 237, 245 nm

[0069] (log ε-4.263; 4.311; 4.136)

[0070] Infrared absorption spectrum (KBr): υOH 3415, υCH 2965, υC═O1730, υCOO⁻1575 cm⁻¹.

[0071]¹H-NMR spectrum (D₂O, δ, ppm): 0.86, d, 3H (2-CH₃); 5.92, dd,J=10.0 and 5.4 Hz, 1H (3-H); 5.99, d, J=10.0 Hz, 1H (4-H); 5.52, br 1H(5-H); 4.24, m 1H (6-H); 5.34, br, 1H (8-H); 4.06, m, 1H(β-H), 3.65, m,1H (δ-H); 1.05, d, 3H (2′-CH₃); 0.82, t, 3H (4′-H₃).

[0072]¹³C-NMR spectrum (D₂O, δ, ppm): 15.3, q (2-CH₃); 139.5, d (C-3);129.5, d, (C-4); 138.1, s (C-4a), 127.7, d (C-5); 66.6, d (C-6); 70.1, d(C-8); 182.6 s (COO⁻); 72.6. d (C-β); 73.0, d (C-δ); 182.0, s (C-1′)18.8; q (2′-CH₃); 13.7, q (C-4′).

[0073] Positive FAB mass spectrum (characteristic ions):

[0074] [M+Na]⁺ 469; [M+H]⁺ 447.

[0075] Negative FAB mass spectrum (characteristic ions):

[0076] [M−H]⁻ 445, [M−Na]⁻ 423, m/z 101 [2-methyl-butyric acid-H]⁻.

Example 3

[0077] In a laboratory scale fermenter with 5 liters working volume,bioconversion culture medium MU/4 was prepared as described in Example1, although it was loaded up to 4.5 liters, the composition of theculture medium was calculated to 5 liters. Then it was sterilized for 45min at 121° C. and inoculated with 500 ml of the inoculum culture madeaccording to Example 1. The fermentation was carried out at 25° C. bythe application of a stirring rate of 300 rpm and an aeration rate of 50liters/hour for 4 days. After 5 g compactin substrate feeding to theculture the bioconversion was carried out according to the Example 2.

[0078] After finishing the bioconversion, the 4.9 liters broth, whichcontained 660 μg/ml pravastatin, was filtered and the separated myceliumwas washed by suspension in 2×1 liter deionized water. The pH of thecombined 5.6 liters filtrate of the broth was adjusted by 20% sulfuricacid to 3.5-3.7, then the acidic filtrate was stirred with 2750 ml ethylacetate for 30 min. Subsequently, the phases are separated. The aqueousphase was extracted again with 2×1375 ml ethyl acetate. 470 ml deionizedwater was added to the combined 4740 ml ethyl acetate extract, then thepH of the aqueous ethyl acetate mixture was adjusted to 7.5-8.0 by 1Msodium hydroxide. After 20 min stirring the phases were separated, thenthe ethyl acetate extract was extracted with 2×235 ml deionized water asdescribed above. Then the combined weakly alkaline aqueous solution of1080 ml volume was concentrated in vacuum to 280 ml volume. Theconcentrated aqueous solution was layered on a chromatographic column(ratio of height:diameter=6.5) filled with 280 ml Diaion HP-20(Mitsubishi Co., Japan) non-ionic resin. The adsorption on the columnwas carried out with a flow rate of 250-300 ml/hour, then the column waswashed with 840 ml deionized water. Subsequently, the column was elutedin the following order with 800 ml 5%, 1000 ml 10%, 500 ml 15% and 500ml 20% acetone-containing water. In the course of the elution 50 mlfractions were collected, which were analyzed by the TLC method given inthe Example 2. Fractions containing pravastatin as the main componentwere combined and the obtained solution was concentrated in vacuum to260 ml volume. The concentrated aqueous solution was chromatographed ona column containing Diaion HP-20 resin in 260 ml volume. After theadsorption of pravastatin the column was washed with 790 ml deionizedwater, then eluted with aqueous acetone solutions in 260-260 ml portionsgradually increasing the acetone content as follows: 2.5, 5.0, 7.5,10.0, 12.5, 15.0 and 20.0%. In the course of the column chromatography25 ml fractions were collected, and the pravastatin content of thefractions was analyzed as given before. Fractions containing pravastatinas the single component by TLC were combined and evaporated in vacuum.Subsequently, 0.3 g charcoal was added to the concentrated aqueoussolution (about 30 ml) and pravastatin was clarified at room temperaturefor 30 min. Then the charcoal was removed by filtration from thesolution and the filtrate was lyophilized. In this way 1.62 gpravastatin was obtained in lyophilized form.

Example 4

[0079] From the slant culture of the Mortierella maculata nov. spec.E-97 [NCAIM(P)F 001266] strain cultivated for 10-12 days, a sporesuspension was prepared with 5 ml sterile 0.9% sodium chloride solution,and this suspension was used to inoculate 500 ml VHIG inoculum mediumbeing sterilized in 3000 ml Erlenmeyer flask.

[0080] Composition of the medium VHIG: glucose  30 g meat extract   8 gyeast extract   1 g Tween-80 (polyoxyethylene (20) sorbitan monooleate)0.5 g in 1000 ml tap water.

[0081] Before the sterilization the pH of the medium was adjusted to 7.0and the sterilization was carried out at 121° C. for 25 min. The culturewas cultivated for 3 days on a rotary shaker (250 rpm, amplitude 2.5cm), then the obtained inoculum culture was used to inoculate alaboratory scale fermenter containing bioconversion culture medium PK in5 liters working volume.

[0082] Composition of the medium PK: glucose  40 g peptone   5 g soybeanmeal  20 g K₂HPO₄   2 g KH₂PO₄   1 g NaNO₃   2 g KCl 0.5 g in 1000 mltap water.

[0083] Before the sterilization the pH of the medium is adjusted to 7.0.After the inoculation, cultivation, the substrate feeding andbioconversion were carried out according to Example 2, then thepravastatin was isolated from the broth in which its concentration was650 μg/ml at the end of the fermentation.

[0084] Finishing the fermentation, the pH of the 4.9 liters brothcontaining 650 μg/ml pravastatin was adjusted under continuous stirringwith 2M sodium hydroxide to 9.5-10.0, then after one hour stirring thepH is adjusted to 3.5-3.7 with 20% sulfuric acid. Subsequently, theacidic solution was extracted with 2.45 liters ethyl acetate. The phasesare separated, and with centrifugation a clear extract was prepared fromthe emulsified organic phase. The broth was extracted again with 2×1.22liters ethyl acetate by the method given above. The ethyl acetateextracts were combined and 0.4 liters deionized water were added, thenthe pH of the mixture was adjusted to 8.0-8.5 with 1M sodium hydroxide.Phases were separated, and the ethyl acetate phase was extracted with2×0.2 liters deionized water of pH 8.0-8.5 as given above. The pH of thecombined pravastatin containing weakly alkaline aqueous solution wasadjusted under stirring with a 20% sulfuric acid solution to 3.5-3.7.The acidic solution obtained was extracted with 4×0.2 liters ethylacetate. The combined ethyl acetate extracts are washed with 2×0.2liters deionized water, then 150 mole % dibenzylamine—calculated for thepravastatin content measured by HPLC—was added into the ethyl acetatesolution. The ethyl acetate solution was concentrated in vacuum to 0.2liters volume. Further 20 mole % dibenzylamine was added to theconcentrate obtained, and the precipitated solution was kept overnightat 0-5° C. The precipitated pravastatin dibenzylamine salt was filtered,then the precipitate was washed on the filter with cold ethyl acetateand then two times with n-hexane, and finally it is dried in vacuum at40-50° C. The crude product obtained (3.9 g) was dissolved in 100 mlmethanol at room temperature, then the solution was clarified by 0.45 gcharcoal. Thereafter the methyl alcoholic filtrate is concentrated invacuum. The evaporated residue was dissolved in 120 ml acetone at anexternal temperature of 62-66° C., then the solution was cooled to roomtemperature. Subsequently, the recrystallization was continued overnightat 0-5° C. Precipitated crystals were filtered, then the crystals werewashed on the filter two times with cold acetone and two times withn-hexane. The recrystallized pravastatin dibenzylamine salt wassuspended in the mixture of 160 ml isobutyl acetate and 80 ml deionizedwater. Subsequently, sodium hydroxide was added in an equivalent amountinto the suspension under stirring. After the disappearance of thesuspension the phases were separated and the pravastatin containingaqueous solution was washed with 2×30 ml isobutyl acetate. The aqueoussolution obtained was clarified with charcoal. Then the aqueous filtratewas concentrated to about 20 ml volume. The aqueous solution obtainedwas loaded on a chromatographic column (height:diameter=22) filled with0.4 liters Sephadex LH-20 gel (supplier: Pharmacia, Sweden). In thecourse of the chromatography deionized water was used as the eluent, and20 ml fractions were collected. Fractions were analyzed by TLC, thenthose containing pravastatin also by HPLC using the methods describedabove. Fractions containing pure pravastatin were combined andlyophilized. In this way 1.75 g pravastatin was obtained, the purity ofwhich is higher than 99.5% by HPLC.

Example 5

[0085] A spore suspension was prepared from the slant culture of theMortierella maculata n. spec. E-97 [NCAIM(P)F 001266] strain cultivatedfor 10-12 days with 5 ml sterile 0.9% sodium chloride solution, and then500 ml inoculum medium was inoculated with it as described in Example 4.In a laboratory scale fermenter with 5 liters working volumebioconversion culture medium PC/4 is sterilized for 45 min at 121° C.and then inoculated with the seed culture.

[0086] Composition of the medium PC/4: malt extract 5.0% soybean meal1.0% peptone 1.0% corn steep liquor 1.0% MgSO₄ × 7 H₂O 0.1% in 1000 mltap water.

[0087] Before the sterilization the pH of the medium is adjusted to 7.0.After the inoculation, the cultivation and substrate feeding werecarried out according to the Example 2, and then 5.1 liters broth with aconcentration of 610 μg/ml pravastatin was obtained.

[0088] From the broth 3.7 g pravastatin dibenzylamine salt crude productwas produced by the method given in Example 4, from which afterrecrystallization 2.9 g pravastatin dibenzylamine salt was obtained. Therecrystallized pravastatin dibenzylamine salt was suspended in 45 mlethanol, then under stirring 110 mole % sodium hydroxide was added bythe feeding of 1M ethanolic sodium hydroxide solution. Stirring of thesolution is continued for half an hour, then 0.3 g charcoal was addedinto it and stirred for another half an hour. The solution was filtered,and the filtrate was concentrated to 15 ml. Then 60 ml acetone was addedto the concentrate at 56-60° C. The solution obtained was cooled to roomtemperature, then kept overnight at +5° C. Subsequently, the precipitatewas filtered, then washed with 2×20 ml acetone, 2×20 ml ethyl acetateand 2×20 ml n-hexane, and finally dried in vacuum. The resulting 1.7 gcrude pravastatin was dissolved in ethanol, then clarified with charcoaland crystallized from an ethanol-ethyl acetate mixture. In this way 1.54g pravastatin was obtained that was identical with the product ofExample 2.

Example 6

[0089] As described in Example 4, from the slant culture of theMortierella maculata n. spec. E-97 [NCAIM(P)F 001266] strain cultivatedfor 7-10 days, a 500 ml inoculum medium MI sterilized in a 3000 mlErlenmeyer flask was inoculated and incubated at 28° C. for 3 days on arotary shaker.

[0090] Composition of the medium MI: glucose   40 g casein   5 g KCl 0.5 g NaNO₃   3 g KH₂PO₄   2 g MgSO₄ × 7H₂O  0.5 g FeSO₄ × 7H₂O 0.01 gin 1000 ml tap water.

[0091] Before the sterilization the pH of the medium is adjusted to 6.0and the sterilization is carried out at 121° C. for 35 min. The seedculture obtained is inoculated into 5 liters bioconversion medium P12sterilized in a fermenter.

[0092] Composition of the medium P12: glucose  10 g malt extract  50 gyeast extract   5 g corn steep liquor   5 g MgSO₄ × 7H₂O   1 g Tween-800.5 g in 1000 ml tap water.

[0093] Before the sterilization the pH of the medium is adjusted to 7.0,then the sterilization was carried out at 121° C. for 45 min. Thefermentation, substrate feeding and bioconversion were carried outaccording to the Example 2. After finishing the bioconversion thepravastatin formed in the concentration of 620 μg/ml was isolated asfollows:

[0094] The pH of 5.15 liters broth containing 620 μg/ml pravastatin wasadjusted with 2M sodium hydroxide to 9.5 value then stirred at roomtemperature for 1 hour. The broth was filtered and the mycelium waswashed with suspension in 1×2 liters and then 1×0.5 liters water.Filtrates are combined and the pH of the aqueous solution was adjustedwith 20% sulfuric acid to 3.7 value and extracted with 2.5 liters thenwith 1.5 liters ethyl acetate. The ethyl acetate extracts were combined,washed with 2×0.5 liters water and 1.95 g dicyclohexylamine was added.The ethyl acetate extract was concentrated at 40° C. to 200 ml underreduced pressure, and 0.195 g dicyclohexylamine was added again into theconcentrate, which was then stirred at 15° C. for 6 hours. Theprecipitated crystalline material was filtered, washed with 20 ml andwith 15 ml ethyl acetate and dried at 40° C. In this way 3.51 g crudeproduct was obtained. After the recrystallization of the crude productin an acetone-ethanol mixture, 3.05 g of pravastatin dicyclohexylaminesalt was obtained (melting point: 162-168° C.), which was converted topravastatin according to the Example 5.

Example 7

[0095] The fermentation, substrate feeding and bioconversion werecarried out with the Mortierella maculata n. spec. E-97 [NCAIM(P)F001266] strain as described in Example 2. Pravastatin obtained as aresult of the bioconversion is isolated from the broth as follows.

[0096] 5 liters culture broth containing in concentration 650 μg/mlpravastatin was filtered on a filter cloth. The mycelium of the mold wasstirred in 2 liters 0.1 M sodium hydroxide solution for an hour, thenfiltered. The two filtrates were combined and the pH was adjusted with15% sulfuric acid to 3.5-4.0. Subsequently, the solution was extractedwith 2×1.8 liters ethyl acetate. The combined ethyl acetate phases werewashed with 800 ml water. Then 400 ml deionized water was added and thepH of the mixture is adjusted by 1M sodium hydroxide to a 8.0-8.5 value.The mixture was stirred for 15 minutes, then the phases were separated.300 ml water was added to the ethyl acetate phase and the pH areadjusted to 8.0-8.5. After stirring for 15 minutes the phases wereseparated. 300 ml water was added again to the ethyl acetate phase andthe pH was adjusted to 8.0-9.5. Then the mixture was stirred for 15 min.The two phases were separated again. All aqueous phases were combinedand the pH are adjusted with 15% sulfuric acid to 3.5-4.0, thenextracted with 3×300 ml ethyl acetate. The combined ethyl acetateextracts were washed with 150 ml water, dried with anhydrous sodiumsulfate, and filtered. Then 150 mole % dioctylamine—calculated for thepravastatin content—was added to the ethyl acetate extract. The ethylacetate was evaporated to about 1/10 volume and acetone was added untilprecipitation. The mixture was kept at +5° C. overnight. The precipitatewas filtered on a G-4 filter, washed with 20 ml acetone and then with 20ml n-hexane and dried in vacuum at room temperature. The 3.3 g crudepravastatin dioctylamine salt obtained was recrystallized from 20 mlacetone resulting in 2.7 g pure pravastatin dioctylamine salt. Meltingpoint: 143-146° C. The pravastatin dioctylamine salt was converted topravastatin with the method given in Example 5.

Example 8

[0097] By the development of the hydroxylation ability of Mortierellamaculata n. spec. E-97 strain isolated from natural habitat, which isable to 6β-hydroxylate compactin, in the mutation-selection and enzymeinduction experiments discussed in detail below, Mortierella maculata n.sp. E-97/15/13 [NCAIM(P)F 001267] mutant strain was produced.

[0098]Mortierella maculata n. sp. E-97 [NCAIM(P)F 001266] strainisolated by us was cultivated on MS slant agar medium at 28° C. for 7days.

[0099] Composition of agar medium MS: glucose  4 g malt extract 10 gyeast extract  4 g agar 20 g in 1000 ml distilled water.

[0100] Spores were washed off from the slant cultures by 5 ml 0.9%sodium chloride solution, then after transferring the spore suspensioninto a sterile Petri dish it was irradiated by ultraviolet light for 1minute. Subsequently, N-methyl-N′-nitro-N-nitrosoguanidine was added tothe spore suspension in the final concentration of 2000 μg/ml. Then thesuspension was transferred into a 100 ml Erlenmeyer flask and it wasshaken at 28° C. with a rate of 150 rpm for 20 min. Subsequently, thespores were sedimented by centrifugation with a rate of 4000 rpm for 10min, then suspended in sterile 0.9% sodium chloride solution. Thesuspension was spread on an agar plate MU-VB containing 10 μg/ml benomyland 1% defibrillated blood.

[0101] Composition of agar medium MU-VB: glucose  40 g asparagine   2 gpeptone 2.5 g potassium dihydrogen phosphate 0.5 g agar  20 g

[0102] in 990 ml distilled water; after sterilization the medium wascompleted with 10 ml bovine blood and 10 mg benomyl.

[0103] The agar plates were incubated at 28° C. for 7 days, then thegrown colonies were transferred by random selection into test tubescontaining agar medium PS.

[0104] Composition of agar medium PS: glucose 40 g mycological peptone10 g agar 15 g in 1000 ml distilled water.

[0105] Before sterilization the pH of the medium is adjusted to 5.6-5.7value. The sterilization is carried out at 121° C. for 20 min.

[0106] Slant cultures were incubated at 28° C. for 12 days, and theirpravastatin productivity was tested in shaken flask experiments asdescribed in Example 1. Mortierella maculata n. sp. E-97/15/13 mutantstrain was selected by this method, which yielded pravastatin exceeding60% conversion rate from the applied compactin acid sodium saltsubstrate being in the concentration of 1000 μg/ml.

[0107] The hydroxylase enzyme of Mortierella maculata n. sp. E-97/15/13strain was induced by the cultivation on MU-VB agar medium containing100 μg/ml 8-de-(2-methyl-butyryl) compactin and/or compactin. Afterrandom selection of the grown colonies they were transferred intoinducer containing MU-VB slants. Pravastatin productivity of the grownslant cultures was examined by the method written in the Example 1 withthe difference that the compactin substrate feeding in the quantity of500 μg/ml was carried out from the 4th day of the fermentation forfurther 11 days and the compactin sodium substrate was added graduallyduring the twelve days converted completely to pravastatin. By the endof the bioconversion carried out in 50 shake flask cultures from 30 gcompactin sodium substrate the formation of 18.5 g pravastatin wasmeasured by HPLC. Recovery of the pravastatin from the combinedfermentation broths was carried out according to the following method.

[0108] After finishing the fermentation the pH of 5.5 liters broth witha pravastatin concentration of 3360 μg/ml was adjusted with 20% sulfuricacid solution to 3.5-3.7. Subsequently, the acidic solution wasextracted by 2.75 liters ethyl acetate. Phases were separated, and aclear extract was prepared by centrifugation from the emulsified organicphase. Broth was extracted two more times with 1.37 liters ethyl acetateas previously described. The combined ethyl acetate extracts were washedwith 2×1.15 liters deionized water, then 150 mole %dibenzylamine—calculated for the pravastatin content measured byHPLC—was added to the ethyl acetate solution. The ethyl acetate solutionwas concentrated in vacuum to about 0.23 liters volume. Further 20 mole% dibenzylamine was added to the concentrate and the precipitatesolution was kept overnight at 0-5° C. Precipitated pravastatin aciddibenzylamine salt was filtered, then the precipitate was washed bysuspending it in cooled ethyl acetate and then two times in n-hexane,finally dried at 40-50° C. in vacuum. The crude product obtained (22.4g) was dissolved in 0.67 liters acetone at 62-66° C. temperature, andthe solution was clarified with 2.2 g charcoal. After the clarificationthe acetone filtrate was concentrated in vacuum to 0.56 liters volume.Crystals precipitated from the concentrate were dissolved again at theabove temperature, then the solution was cooled to room temperature.Subsequently, the recrystallization was continued overnight at 0-5° C.Precipitated crystals were filtered, and washed by suspension two timesin cooled acetone and two times in n-hexane. Recrystallized pravastatinacid dibenzylamine salt was dried in vacuum at 40-50° C. Pravastatinacid dibenzylamine salt obtained (14.8 g) was dissolved at 40-44° C. in740 ml ethyl acetate-ethanol (9:1) mixture, then 110 mole % sodiumhydroxide was added to the solution in form of a 1M ethanolic solution.Stirring of the obtained precipitated solution was continued for half anhour at room temperature, then a complete precipitation was achieved asa result of the application or ice cooling for 1-1.5 hours.Subsequently, the precipitate was filtered and washed with 2×150 mlcooled ethyl acetate and 2×150 ml n-hexane, finally dried in vacuum at40-50° C. The pravastatin obtained was dissolved in ethanol, clarifiedby 1.0 g charcoal, then crystallized from ethanol-ethyl acetate mixture.This way 9.4 g pravastatin was obtained, with physical constantscorresponding to the data given in Example 2.

[0109] Although certain presently preferred embodiments of the inventionhave been described herein, it will be apparent to those skilled in theart to which the invention pertains that variations and modifications ofthe described embodiments may be made without departing from the spiritand scope of the invention. Accordingly, it is intended that theinvention be limited only to the extent required by the appended claimsand the applicable rules of law.

We claim:
 1. A microbial process for the preparation of the compound of formula (I)

from a substrate compound of formula (II),

wherein R stands for an alkali metal or ammonium ion, comprising the steps of a) cultivating a strain of Mortierella maculata filamentous mold species able to 6β-hydroxylate a compound of formula (II) on a nutrient medium containing assimilable carbon and nitrogen sources and mineral salts, b) feeding the substrate to be transformed into the developed culture of Mortierella maculata, c) fermenting the substrate until the end of bioconversion, d) separating the compound of formula (I) from the culture broth, and e) isolating the compound of formula (I).
 2. The process of claim 1, wherein said medium is a nutrient broth.
 3. The process of claim 2, wherein said step of separating the compound of formula (I) from the culture broth is performed by adsorption on an anionic ion exchange resin.
 4. The process of claim 2, wherein said step of separating the compound of formula (I) from the culture broth is performed by extraction with a water immiscible organic solvent, followed by the preparation of its lactone derivative or its secondary amine salt as an intermediate.
 5. The process of claim 2, wherein said step of separating the compound of formula (I) from the culture broth is performed by purification of the alkaline aqueous extract obtained from the organic solvent extract of the fermentation broth with chromatography on a non-ionic adsorbing resin.
 6. The process of claim 1, wherein the strain of Mortierella maculata is the Mortierella maculata n. sp. E-97 strain deposited at the National Collection of Agricultural and Industrial Microorganisms, Budapest, Hungary under accession number NCAIM(P)F
 001266. 7. The process of claim 1, wherein the strain of Mortierella maculata is the Mortierella maculata n. sp. E-97/15/13 strain deposited at the National Collection of Agricultural and Industrial Microorganisms, Budapest, Hungary under accession number NCAIM(P)F
 001267. 8. The process of claim 1, wherein the hydroxylase enzyme of the strain used for the transformation is induced by 8-de(2-methyl-butyryl) compactin or compactin.
 9. The process of claim 2, wherein a compound of formula (II) as a substrate is added into the culture parallel with the feeding step, and wherein the feeding step depends on the pH of the culture and its quantity is 0.5-1.0% related to the volume of the broth.
 10. The process of claim 1, wherein the fermentation step is carried out on a medium containing a carbon source selected from the group consisting of glucose, fructose and glycerine.
 11. The process of claim 1, wherein the fermentation step is carried out on a medium containing a nitrogen source selected from the group of consisting of soybean meal, peptone, casein, yeast extract and meat extract.
 12. The process of claim 2, wherein the compound of formula (I) formed during the bioconversion is separated from the culture broth by adsorption from the filtrate of the broth and from the washing water of the mycelium on an anion exchange resin, eluting the compound of formula (I) from the resin, transforming the compound of formula (I) completely to its lactone form, isolating the lactone derivative, hydrolyzing the lactone derivative by sodium hydroxide, and desalting the compound of formula (I) on a non-ionic adsorption resin.
 13. The process of claim 12, wherein said anion exchange resin has quaternary ammonium active groups carrying polystyrene-divinylbenzene skeleton, is used for the separation of the compound of formula (I) from the filtrate of the broth.
 14. The process of claim 4, wherein the compound of formula (I) formed during the bioconversion is extracted in acid form from the broth, the broth having been acidified to the pH of 3.5-3.7, or from filtrate of the broth by a water-immiscible organic solvent.
 15. The process of claim 14 wherein said water-immiscible organic solvent is ethyl acetate.
 16. The process of claim 14 wherein said water immiscible organic solvent is isobutyl acetate.
 17. The process of claim 5, wherein said compound of formula (I) is extracted in sodium salt form from the organic solvent by aqueous sodium hydroxide solution, and purified on a non-ionic adsorption resin.
 18. The process of claim 14, wherein said compound of formula (I) is precipitated from the extract in crystalline form with a secondary amine containing alkyl-, cycloalkyl-, aralkyl- or aryl-substituents.
 19. The process of claim 18, wherein the crystalline secondary amine salt is suspended in a mixture of a 1-4 carbon atom-containing alkyl ester of acetic acid and water; an equivalent quantity of sodium hydroxide is added in aqueous solution to the suspension such that an organic phase and an aqueous phase are formed; the organic and aqueous phases are separated; the aqueous phase is washed with isobutyl acetate, then clarified with activated carbon; and the aqueous solution is lyophilized.
 20. The process of claim 19, wherein said alkyl ester is isobutyl ester.
 21. The process of claim 18, wherein the crystalline secondary amine salt is suspended in a 1-4 carbon atom-containing alcohol; from the suspension a solution of the compound of formula (I) is prepared by adding an ethanolic solution of sodium hydroxide; and the compound of formula (I) is precipitated from the solution by acetone.
 22. The process of claim 21, wherein said 1-4 carbon atom-containing alcohol is ethanol.
 23. The process of claim 18, wherein the crystalline secondary amine salt is dissolved in a mixture of a 1-4 carbon atom-containing alkyl ester of a 1-4 carbon atom-containing alkane carboxylic acid and a 1-4 carbon atom-containing alcohol; and from the solution the compound of formula (I) is precipitated in crystalline form by adding sodium hydroxide.
 24. The process of claim 23, wherein said mixture is an ethyl acetate-ethanol mixture.
 25. The process of claim 19, wherein pravastatin is isolated from the fermentation broth via the dibenzylamine salt of the acid form of the compound of formula (I).
 26. The process of claim 19, wherein the acid derivative of the compound of formula (I) is purified through its dicyclohexylamine salt.
 27. The process of claim 19, wherein the acid derivative of the compound of formula (I) is purified through its dioctylamine salt.
 28. The process of claim 19, wherein the compound of formula (I) is purified to at least 99.5%, as measured by HPLC, using gel chromatography.
 29. The process of claim 1, wherein said strain of Mortierella maculata is cultivated at about 25° to about 30° C.
 30. A biologically pure culture of the Mortierella maculata n. sp. E-97 strain deposited at the National Collection of Agricultural and Industrial Microorganisms, Budapest, Hungary under accession number NCAIM(P)F
 001266. 31. A biologically pure culture of the Mortierella maculata n. sp. E-97/15/13 strain deposited at the National Collection of Agricultural and Industrial Microorganisms, Budapest, Hungary under accession number NCAIM(P)F
 001267. 32. A Mortierella culture able to 6β-hydroxylate a compound of formula (II)

wherein R stands for an alkali metal or ammonium ion, consisting essentially of a novel strain Mortierella maculata n. sp. E-97 deposited at the National Collection of Agricultural and Industrial Microorganisms, Budapest, Hungary under accession number NCAIM(P)F
 001266. 33. A Mortierella culture able to 6β-hydroxylate a compound of formula (II)

wherein R stands for an alkali metal or ammonium ion, consisting essentially of a novel strain Mortierella maculata n. sp. E-97/15/13 deposited at the National Collection of Agricultural and Industrial Microorganisms, Budapest, Hungary under accession number NCAIM(P)F
 001267. 